Bone fractures pose a significant problem for veterans who suffered from traumatic injury in the line of military duty or from prevalent osteoporotic diseases as a civilian. Intermittent (or daily) administration of parathyroid hormone (PTH) is the only FDA-approved pharmaceutics that produces osteoanabolism to treat osteoporosis. The osteoanabolic action of PTH is based on the ability of the hormone to alter systemic and local factors that promote osteoblast (OB) activity before its stimulation of osteoclast (OCL) activity catches up, creating an ?anabolic window? of positive effects on bone mass and structure to restore mechanical integrity of the bone. Intermittent PTH has also been shown to promote chondrogenesis in calluses at early stages of fracture repair in preclinical models. Several clinical trials also demonstrated osteoanabolism of intermittent PTH at fracture sites. Its dosing is, however, limited to a low level and a short duration due to potential adverse effects -- hypercalcemia and induction of osteosarcoma. Our proposal aims to delineate mechanisms underlying the osteoanabolic actions and the hypercalcemic effects of PTH in order to devise new strategies to enhance PTH therapy. Raising [Ca] activates the extracellular calcium-sensing receptors (CaSRs) in chondrocytes and OBs to promote their survival and differentiation and in OCLs to inhibit their survival and bone-resorbing functions. We postulate that the hypercalcemic effect of intermittent PTH is essential for the production of osteoanabolism. A class of allosteric CaSR agonist (or calcimimetics) has been used clinically to treat hyperparathyroidism and hypercalcemia by potentiating extracellular Ca-induced inhibition of PTH secretion in parathyroid cells (PTCs). In this new grant application, we postulate that this compound, when co-injected with intermittent PTH, will not only subside the hypercalcemic side effects of PTH, but also synergize the effects of PTH and enhance skeletal anabolism by activating the CaSRs in chondrocytes, OBs, and OCLs directly. We hypothesize that a concurrent calcimimetic treatment promotes chondro-to-osteo transition and enhances osteoanabolism of intermittent PTH1-34 by activating the CaSRs in chondrocytes, OBs, and/or OCLs to increase fracture repair capacity and concurrently rehabilitate other weakened skeletons. Aim 1 will determine whether simultaneously activating CaSRs in PTCs, chondrocytes, OBs, and OCLs by systemic co-administration of R568 with PTH1-34 abrogates hypercalcemia and produces more robust osteoanabolism than administration of PTH1-34 or R568 alone to (a) speed up structural and functional recovery of the bone subjected to a unilateral tibial mid-shaft fracture procedure and (b) to increase structural and mechanical strength of the contralateral bone in adult and aging mice. Aim 2: determine (a) whether the expression of chondrocytic CaSR is required for fracture healing and the anabolic effects of PTH1-34/R568 on promoting chondrocyte differentiation and their transition into OBs by testing the effects of chondrocyte-specific CaSR KO on the fractured calluses in the presence or absence of those drugs; and (b) whether the expression and activation of CaSRs in OBs and/or OCLs are essential for the osteoanabolic effects of the PTH1-34/R568 treatment by comparing the effects of this regimen on fractured and uninjured skeleton in mice with their CaSR ablated in OBs or OCLs, respectively. Aim 3 will delineate the cell-autonomous mechanisms underlying the effects of PTH1-34/R568 on the chondro-to-osteo transition by examining the effects of the compounds on the proliferation, survival, differentiation, mineralizing functions, and signaling responses in cultured callus chondrocytes lacking CaSR and/or PTH1R. Our preliminary studies showed that co-injections of intermittent PTH with a calcimimetic, NPS-R568, (i) prevent hypercalcemia, (ii) enhance anabolic effects on both trabecular and cortical bone, (iii) promote healing of tibial bone fractures, and (iv) enhance transdifferentiation of callus chondrocytes into osteoblasts in mice. Successful completion of this proposal will establish a novel and feasible regimen to rehabilitate fractured and osteoporotic bones in large populations of VA patients.